EXAM3 : data interpretation Flashcards
what are the steps in STR genotyping / interpretation
- peak detection/interpretation thresholds
- peak sizing
- DNA size (allele call)
- DNA fragment analysis
what does internal size standard do?
it runs along with the sample and it shows the size vs time of DNA fragments
___ is the bin defined around each allele
+/- 0.5
what is allele call?
determined by comparison of
peak size (bp) to allelic ladder
allele peak sizes run under the
same electrophoretic conditions
what is the detection threshold ?
50-200 RFU
why do we use thresholds?
to separate signal from noise (is the peak real or not)
analytical RFU ____
50
Interpretation RFU
150 RFU
what is signal peak height measured in ? what is it related to?
Relative Fluorescence Units (RFU)
- related to the amount of DNA present in the sample loaded onto the analysis instrument
what 3 issues impact mixture interpretation?
1.) peak detection threshold
2.) peak height ratio
3.) stutter percentage
biological artifact vs technology artifact issues
B: stutter, “N” peak, Off ladder, Tri-Alleles
T: Pull up, Dye blobs, air bubbles, urea crystals/particles, and voltage peaks
what happens during allelic drop-outs?
degradation and inhibition of the primer binding site mutation
what are the data interpretation issues
- artifact peaks vs allele peaks
- allelic drop outs
- mixtures
- mutations
what are stutter products ?
peaks that show up primarily one repeat less than the true allele
how does stutter happen?
a result of strand slippage during DNA synthesis
which repeat regions generate more stutter?
longer
where is stutter less pronounced ?
larger repeat unit sizes
true or false: each successive stutter product is less intense
true
how much of the true allele in tetranucleotides repeats STR loci
5-15%
(N-4) stutter product is …
Deletion - caused by slippage on the copied BOTTOM strand
(N+4) stutter product is …
Insertion - caused by slippage of the copying TOP strand
In strand slippage, the lower incorporation rate allows what?
more opportunity for the DNA strands to breathe apart during PCR (same mechanism as mutation)
Taq Polymerase - stutter peaks
50-60 nt/second
Stoffel fragment - stutter peaks
5-10 nt/second
Is a “stutter” the same size as an allele ?
YES !
what is the average stutter in D3S1358/ vWA?
7%
what is the average stutter for TPOX?
3%
percent stutter tends to increase with __
allele length
the proportion of stutter to main allele is …
generall reproducible within a locus and even for a given allele length
alleles with longer core repeat unit regions generally have _____ proportion of stutter
increase
alleles with interrupted core repeat regions tend to have ___ proportion of stutter
less
what are the strategies for stutter reduction?
- cosolvents: osmolytes
- cycling parameters
- mutants of taq polymerase
In non-template addition what does taq polymerase do?
it will often add an extra nucleotide to the end of a PCR product , often “A” (adenlyation)
why is non-template addition dependent on the 5’-end of the reverse primer?
it is dependent on this end so that a “G” can be put at the end of a primer to promote the non-template addition
how can the non-template addition be enhanced?
with extension soak at the end of the PCR cycle ( 15-45 min @60 or 72 degrees to give polymerase more time)
excess amounts of DNA template in the PCR reaction can result in…
incomplete adenylation (not enough polymerase to go around)
why do we NOT want a mixture of (+/- A peaks)
to avoid split peaks
What observations indicate that a peak in an electropherogram is a
spike as opposed to DNA?
sharp peak, that shows up in all of the panels (spike), where the allele is more curved
Is it better to prevent or promote non-template addition? Explain your
reasons?
promote, to get it to A+ , we want it to be A+!!
What steps are typically taken to confirm the presence of an off-
ladder allele?
does not match up with allelic ladder, meaning it doesn’t show up in the ladder
** if it is off then you would run it again, and again
- sometimes there is a temperature difference in your sample vs ladder which would mess up with alignment
- if you keep seeing it then it is off ladder
